B. Protein Blotting

A general protocol for sample preparation.

Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.

Western Blot Reprobing Protocol

Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.

(Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.

Wash membrane again four times for 5 min each in TBST.

The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

Source / Purification

Background

GFI1b and its homolog GFI1 are transcriptional repressors and important regulators of erythroid and megakaryocytic development and differentiation (1,2). GFI1b negatively regulates transcription by recruiting chromatin regulatory proteins including CoREST, the histone demethylase LSD1 and HDACs 1 and 2, which associate with GFI1b via its SNAG repression domain (3). GFI1b has also been shown to control the differentiation of erythroid and megakaryocytic progenitors by regulating TGF-β signaling at the bipotent progenitor stage (4). Inactivation of GFI1b in mice leads to embryonic lethality due to failure to produce functional erythrocytes and megakaryocytes (2). The GFI1b gene locus can be autoregulated by binding to its own promoter in hematopoietic cells, likely through interacting with GATA-1, another transcription factor essential for erythroid and megakaryocytic development (5). Mutations in GFI1b are implicated in various leukemias (6) and GFI1b has been found in a complex with GATA-1 and SUZ12 on repressed genes in erythroleukemia cells (7).